SUMMARY To date, fundamental understanding of which features of odorants are decoded by the brain, and how information about these features is channeled through the olfactory system is still lacking. Odorants are sensed by the olfactory sensory neurons (OSNs) in the olfactory epithelium expressing specialized odorant receptors (ORs). Each OSN expresses one OR gene out of a species-dependent complement of hundreds of OR genes. OSN axons expressing the same OR type converge onto the same glomerulus on the olfactory bulb (OB) surface, forming a 2D map which is approximately stereotypical across individuals. Mitral/tufted cells (MTCs), the principal neurons of the OB are driven by inputs from glomeruli, as well as lateral and top-down signals. MTCs send their axons to higher olfactory processing centers, forming what is commonly assumed to be, highly distributed and largely random projection patterns. Computational models of olfactory processing are sensitive to the structure of the MTC projectome, with different models relying on different statistics of connections. Determining the structure of MTC connectivity is therefore of utmost importance for understanding the computational principles underlying olfactory information processing. However, to date, the structure of these projections across individuals remains uncharted territory, and information on the statistics of projections for ensembles of single MT neurons per individual is very limited, especially, in mammals. This is due to the low yield of imaging-based anatomical reconstruction strategies via sparse labeling of a small number of individual neurons per brain. To understand the logic and specificity of the MTC projectome, in this project, we will leverage the high throughput of state-of-the-art sequencing technologies, such as fluorescence in situ sequencing (FISSEQ) and a novel RNA barcoding sequencing-based method (MAPseq) in conjunction with in vivo functional imaging, modern computational technologies and theoretical tools. Preliminary data comprising of the brain-wide projections of hundreds of individual neurons supports the existence of specialized, non-random projection motifs that can be compared between animals. We will further investigate the structure of the brain-wide MTC projections and relate it to the MTC responses to large sets of odorants. We will share this data with the broader olfaction community and incorporate it into a computational network model of olfactory processing. The Specific Aims (SAs) of this project are: SA1. To determine the logic and specificity of individual mitral and tufted cells projections across the major target brain regions of the olfactory bulb. SA2. To investigate the structure of mitral and tufted cells' projectome within individual OB target brain regions. SA3. To understand the relationship between the bulb projectome and the odor responses of mitral and tufted cells.